Sperm capacitation is a poorly understood series of molecular processes that occur in vivo in the female genital tract, and is absolutely essential to a sperm's ability to fertilize an egg. A key component of capacitation is the hyperpolarization of the sperm plasma membrane, but the molecular mechanism underlying hyperpolarization was unknown. We previously showed by using a SLO3 knock-out (SLO3 -/-) mouse that the SLO3 potassium channel is the main ion channel responsible for the hyperpolarization that occurs during sperm capacitation and that the SLO3 -/- is male infertile. We also demonstrated that sperm from SLO3 -/- mice have major deficits in sperm motility as well as in the Acrosome Reaction (AR). Since SLO3 channels are the major determinant of changes in membrane potential (Em) that occur during capacitation, it should now be possible to reveal how the components of the capacitating media trigger the activation of SLO3 channels and how SLO3 channel activation affects other signaling pathways that are active during capacitation and the AR. Since SLO3 -/- sperm have impaired motility and AR, and both events require increases in [Ca2+]i, we will investigate how SLO3 channel activation and subsequent changes in Em affect the [Ca2+]i changes that occur during capacitation and the AR. The overall objective of this proposal is to reveal the role of sperm membrane potential (Em) changes in capacitation and the AR. The SLO3 -/- mouse now represents an invaluable tool which permits us to gain greater experimental control over sperm Em and will reveal the importance of Em in each element of sperm physiology examined. The fact that SLO3 channels are essential to fertilization also suggests the possibility that genetic variation within this gene accounts for differences in fertility among male individuals. SLO3 channels also represent an ideal target for designing and testing blockers specific for this essential channel that can be used as contraceptive drugs. Finally, knowledge of the electrical properties of sperm and the essential aspects of the external ionic environment may have important implications for improving the efficiency of clinical IVF procedures. PUBLIC HEALTH RELEVANCE: The slo3 gene encodes a high conductance K+ channel found only in mammals and expressed exclusively in male germ cells. Using the SLO3 knock out mouse line and a variety of molecular, physiological, and transgenic techniques we will reveal voltage-sensitive processes that are essential for sperm capacitation and the acrosome reaction, information that may impact the field of in-vitro fertilization and contraception.